Potassium hunting on protein factories

Amazing insights into the location of elusive potassium ions on bacterial ribosomes

Groundbreaking research at the new long-wavelength macromolecular crystallography beamline (I23) at Diamond Light Source has for the first time demonstrated the location of potassium ions in bacterial ribosomes. Ribosomes are the protein factories of cells and although they are vital for life, little was known of the sites of metal ions that are crucial for their structure and function. The work recently published in Nature Communications showcases the fantastic applications of the I23 beamline and sheds light on the important role of potassium ions.

>Read more on the Diamond Light Source website

Image: (extract, full image here) 70S ribosome elongation complex (potassium atoms rendered as green spheres).

The interaction between two proteins involved in skin mechanical strength

A research team from the Centro de Investigación del Cáncer of the Universidad de Salamanca has obtained a detailed 3D image of the union between two hemidesmosomal proteins.

The structure of this complex has been unveiled using XALOC beamline techniques, at the ALBA Synchrotron. The results, published in “Structure”, provide insights to understand how these epithelial adhesion structures are formed. Researchers from Centro de Investigación del Cáncer – Instituto de Biología Molecular y Celular del Cáncer of Salamanca, from Centro Universitario de la Defensa in Zaragoza, and from the Netherlands Cancer Institute in Amsterdam have described how two essential proteins interact to each other in order to join epidermis and dermis together. This study reveals at atomic scale how the binding between two hemidesmosomal proteins called integrin α6β4 and BP230 takes place.
Epithelial tissues, such as epidermis, settle on fibrous sheets called basement membrane, formed by extracellular matrix proteins. The junction between epithelia and basement membrane happens through hemidesmosomes, multi-protein complexes located at the membrane of epithelial cells. Integrin α6β4 is an essential protein of the hemidesmosomes, which adheres to proteins of the basement membrane. Inside the cell cytoplasm, plectin and BP230 proteins bind to α6β4 and connect it to the intermediate filaments of the cytoskeleton. Genetic or autoimmune alterations that affect the hemidesmosomal proteins reduce skin resistance and cause diseases such as bullous pemphigoid and various types of epidermolysis bullosa.

>Read more on the ALBA website

Image: Structure of β4(WT)-BP230 complex.

Capturing protein motion at FemtoMAX

Your body contains a large variety of different proteins. They are big, complex molecules with diverse functions, from transporting oxygen in your blood to making your muscles contract.

Many proteins change their shape and move as they perform their task. A research team from the University of Gothenburg recently visited the beamline FemtoMAX to develop a method for studying moving proteins. They use electric fields to stimulate motion of the proteins in a sample while imaging them with the X-ray beam.
To study how proteins move, we need something to nudge them and then image them after they have changed position. Certain proteins are activated by light and in that case, the researchers can hit them with a laser pulse to provoke the motion. However, that is far from always the case. In the method being developed by the Gothenburg team, the proteins are instead subjected to an electric field that make them move.
The field is synchronized to the short, femtosecond scale (10-15 s) X-ray pulses delivered at beamline FemtoMAX. Each X-ray pulse hitting the sample is like taking a photograph using extremely short shutter speed, just like trying to get sharp images of players on a football field. The X-ray pulses at FemtoMAX are short enough to let the researchers capture the instantaneous position of the protein. By varying the time between the electric field and the X-ray pulse they can see different stages of the movement and even put the frames together as a movie of the protein motion.

>Read more on the MAX IV Laboratory website

A new molecule could help put the STING on cancer

The protein STING (stimulator of interferon genes) is a component of the innate immune system. It plays a major role in the immune response to cancer, and abnormal STING signaling has been shown to be associated with certain cancers. Immunomodulatory approaches using agonists to target STING signaling are therefore being investigated as anticancer treatments. However, the compounds in clinical trials typically are injected intratumorally in patients with solid cancers. In this study, researchers discovered a novel STING agonist, known as an amidobenzimidazole (ABZI), which can be given by intravenous injection and could therefore potentially open up its evaluation as a treatment for hard-to-reach cancers. Using x-ray diffraction data collected at the U.S. Department of Energy’s Advanced Photon Source (APS), researchers from GlaxoSmithKline (GSK) investigated ABZI compounds and STING. Their results, published in the journal Nature, may have important implications for anticancer immunotherapy.

STING is a protein that mediates innate immunity, and one function of the STING signaling pathway is in mobilizing an immune response against tumors. STING proteins can be activated by cyclic dinucleotides, small molecules that are made by the cytosolic DNA sensor, cGAS, upon sensing of DNA leaking out of the nucleus as a result of DNA damage, including that which might be associated with cancer development.

>Read more on the Advanced Photon Source at Argonne National Lab.

Figure: X-ray crystal structure of the STING protein bound to one of the new molecules.

Clear view of “Robo” neuronal receptor opens door for new cancer drugs

During brain development, billions of neuron nerve cells must find accurate pathways in the brain in order to form trillions of neuronal circuits enabling us to enjoy cognitive, sensory and emotional wellbeing.

To achieve this remarkable precision, migrating neurons use special protein receptors that sense the environment around them and guide the way so these neurons stay on the right path. In a new study published in Cell, researchers from Bar-Ilan University and Tel Aviv University in Israel, EMBL Grenoble in France and University of Exeter in the UK report on their discovery of the intricate molecular mechanism that allows a key guidance receptor, “Robo”, to react to signals in its environment.

One of the most important protein signaling systems that guide neurons consists of the cell surface receptor “Robo” and its external guidance cue, “Slit”. “Slit and Robo can be identified in virtually all animals with a nervous system, from a 1 mm-long nematode all the way to humans,” explains researcher Yarden Opatowsky, associate professor and head of the Laboratory of Structural Biology at Bar-Ilan University and who led the research.

>Read more on the European Synchrotron website

Image: A surface representation of the crystal structure of the extracellular portion of human Robo2. The yellow region represents the domain where dimerisation takes place. Here, we see it blocked by the other domains, meaning dimerisation cannot take place and that Robo2 is inactivated.
Credit: Y. Opatowsky.

Encapsulation of drugs for new cancer treatments

Research develops hydrogel from silk protein with potential application in photodynamic therapy

Cancer is a set of diseases characterized by uncontrolled multiplication of cells. One of the main methods for treating this disease is chemotherapy, which uses drugs to block the growth of those cells or to destroy them. In this way, most drugs used interfere with mitosis, the cellular mechanism by which new cells are produced. Therefore, both cancerous and healthy cells are affected, leading to several side effects.
Worldwide, considerable effort has been directed at developing new methods that act directly on the target of treatment. This is the case of so-called photodynamic therapy (PDT), a minimally invasive therapeutic procedure that selectively acts on malignant cells.
The procedure involves the administration of a light-sensitive substance, called a photosensitizing agent. When irradiated at specific wavelengths, the photosensitizer releases oxygen in reactive chemical forms that promote the death of malignant cells, infectious agents and the removal of burns.

>Read more on the Brazilian Synchrotron Light Laboratory (LNLS) website

A timely solution for the photosynthetic oxygen evolving clock

XFEL Hub collaboration reveals the intermediates of the photosynthetic water oxidation clock

A large international collaborative effort aided by the XFEL Hub at Diamond Light Source has generated the most detailed time-resolved studies to date of a key protein involved in photosynthesis. The pioneering work, recently published in Nature, shows how photosystem II harnesses light energy to produce oxygen – insights that could direct a next generation of photovoltaic cells. 
>Read more on the Diamond Light Source website

Image: this figure is issued from a video you can watch here.

A two-pronged defense against bacterial self-intoxication

Researchers solved the structure of a bacterial toxin bound to a neutralizing protein, revealing two distinct mechanisms for how the toxin-producing bacteria avoid poisoning themselves.

Microbial communities are of fundamental importance to virtually all natural ecosystems, from the ocean floor to the gastrointestinal tract. Although the term “communities” implies cooperation, scientists now realize that bacterial colonies compete with each other for life-sustaining resources, availing themselves of a variety of strategies to reduce overcrowding. In some cases, they secrete toxins in their fight for survival. Here, researchers studied one such toxin from the bacterium Serratia proteamaculans, various strains of which live inside tree roots or inhabit the digestive tracts of insects and other animals.

Toxin targets cell division

The researchers showed that the toxin, Tre1, targets a bacterial protein, FtsZ, which is analogous to tubulin in human cells. Tubulin molecules are the building blocks of microtubules—long polymers that provide structure and shape to our cells and play an important role in cell division. In bacteria, FtsZ loses the ability to polymerize when attacked by the Tre1 toxin. Instead of dividing, the intoxicated cells grow longer and longer until they eventually split open and die (cellular elongation and lysis).

>Read more on the Advanced Light Source website

Image: Healthy bacteria (left) and bacteria (right) whose cell-division machinery has been disrupted by a toxin newly discovered in some bacterial arsenals.
Credit: Mougous Lab

Understanding the protein responsible for regulating heartbeats

A new research project uses the Canadian Light Source to help researchers understand the protein responsible for regulating heartbeats. Errors in this crucial protein’s structure can lead to potentially deadly arrhythmias, and understanding its structure should help researchers develop treatments. This protein, calmodulin (CaM), regulates the signals that cause the heart to contract and relax in almost all animals with a heartbeat.

“Usually you find some differences between versions of proteins from one species to another,” explains Filip Van Petegem, a professor in the University of British Columbia’s Department of Biochemistry and Molecular Biology. “For calmodulin that’s not the case—it’s so incredibly conserved.”

It also oversees hundreds of different proteins within the body, adjusting a broad array of cellular functions that are as crucial to our survival and health as a steady heartbeat.

>Read more on the Canadian Light Source website

Image: A surface representation of the disease mutant CaM (D95V, red) in complex with the piece of the voltage-gated calcium channel (blue).

Know your ennemy

Light source identifies a key protein interaction during E. coli infection

Escherichia coli is a common source for contaminated water and food products, causing the condition known as gastroenteritis with symptoms that include diarrhea, vomiting, fever, loss of energy, and dehydration. In fact, for children or individuals with weakened immune systems, this bacterial infection in the gut can be life-threatening.

One of the microbes responsible for gastroenteritis, known formally as enteropathogenic E. coli (EPEC), causes infections by directing a pointed, needle-like projection into the human intestinal tract, releasing toxins that make people sick.

“Enteropathogenic E. coli can fire toxic proteins from inside the bacterium right into the cells of your gut lining,” says Dustin Little, a post-doctoral researcher in the Brian Coombes lab at McMaster University’s Department of Biochemistry and Biomedical Sciences.

>Read more on the Canadian Light Source website

Image: Dustin Little and Brian Coombes in the lab.
Credit: Dustin Little. 

SwissFEL makes protein structures visible

Successful pilot experiment on biomolecules at the newest large research facility of PSI

For the development of new medicinal agents, accurate knowledge of biological processes in the body is a prerequisite. Here proteins play a crucial role. At the Paul Scherrer Institute PSI, the X-ray free-electron laser SwissFEL has now, for the first time, directed its strong light onto protein crystals and made their structures visible. The special characteristics of the X-ray laser enable completely novel experiments in which scientists can watch how proteins move and change their shape. The new method, which in Switzerland is only possible at PSI, will in the future aid in the discovery of new drugs.

Less than two years after the X-ray free-electron laser SwissFEL started operations, PSI researchers, together with the Swiss company leadXpro, have successfully completed their first experiment using it to study biological molecules. With that, they have achieved another milestone before this new PSI large research facility becomes available for experiments, at the beginning of 2019, to all users from academia and industry. SwissFEL is one of only five facilities worldwide in which researchers can investigate biological processes in proteins or protein complexes with high-energy X-ray laser light.

>Read more on the SwissFEL website

Image: Michael Hennig (left) and Karol Nass at the experiment station in SwissFEL where their pilot experiment was conducted.
Credit: Paul Scherrer Institute/Mahir Dzambegovic

Year of Engineering I23 Gripper Spotlight

Celebrating the Year of Engineering on Beamline I23

The Year of Engineering (UK) is all about celebrating the world and wonder of the industry, and exploring the wide range of ideas and innovations that Engineering involves. Today, we’re having a look at Diamond’s Beamline I23 – a specially designed instrument for protein crystallography that uses long wavelengths.
There are unique engineering scientific challenges involved in designing a system that will allow researchers to use long wavelengths of Synchrotron radiation effectively. The special cryogenically-cooled sample gripper on I23, is one of the solutions that makes this beamline successful. Learn more about this engineering innovation.

>Read more and watch more videos on the Diamond Light Source website

First European XFEL research results published

High number of X-ray pulses per second reduces time needed for the study of biological structures.

Just days before the first anniversary of the start of European XFEL user operation, the first results based on research performed at the facility have been published. In the journal Nature Communications, the scientists, headed by Prof. Ilme Schlichting from Max-Planck-Institute for Medical Research in Heidelberg, Germany, together with colleagues from Rutgers State University of New Jersey, USA, France, DESY and European XFEL, describe their work using the intense X-ray laser beam to determine the 3D structure of several proteins. They demonstrate, for the first time that, under the conditions used at the time of the experiment an increased number of X-ray pulses per second as produced by the European XFEL can be successfully used to determine the structure of biomolecules. As much faster data collection is therefore possible, the time needed for an experiment could be significantly shortened. The detailed determination of the 3D structure of biomolecules is crucial for providing insights into informing the development of  novel drugs to treat diseases.

Prof. Ilme Schlichting said: “Our work shows that under the conditions used data can be collected at European XFEL at a rate much faster than has ever been previously possible. As the time and cost of experiments decrease, very soon many more researchers will be able to perform experiments at high repetition rate X-ray lasers. Our results are therefore of interest not only tor the fields of biology and medicine, but also physics, chemistry and other disciplines.”

>Read more on the European XFEL website

Image: Guest scientist Tokushi Sato working at the sample chamber of the SPB/SFX instrument.
Credit: European XFEL

Structure reveals mechanism behind periodic paralysis

The results suggest possible drug designs that could provide relief to patients with a genetic disorder that causes them to be overcome suddenly with profound muscle weakness.

A rare genetic disorder called hypokalemic periodic paralysis (hypoPP) causes sudden, profound muscle weakness in people who occasionally exhibit low levels of potassium in their blood, or hypokalemia. When a patient is hypokalemic, hypoPP affects the function of the muscles responsible for skeletal movement. The disease has been known to stem from mutations in certain membrane proteins that channel and regulate the flow of sodium into cells. Exactly how the mutation affects the proteins’ function, however, was not known.

In earlier work, researchers from the Catterall Lab at the University of Washington had solved the structure of a sodium channel called NavAb from a prokaryote (single-celled organism). As a next step, the group decided to see if NavAb could serve as a model for studying the mutations that cause hypoPP in humans (eukaryotes), with the goal of finding a way to prevent or treat this disorder.

A leak in the pipe?

In a resting state, muscle-cell membranes keep potassium ions and sodium ions separated, inside and outside the cell, respectively, creating a voltage across the membrane. A chemical signal from a nerve cell sets off a cascade of events that results in sodium ions flowing into the cell, changing the membrane potential and and ultimately triggering muscle contraction.

>Read more on the Advanced Light Source website

Image: Three states of the voltage-sensing domain (VSD) of a membrane-channel protein. In the normal state, the water-accessible space (magenta) does not extend through the channel, preventing sodium (gray spheres) from passing through. In the disease state, a clear passage allows sodium to leak through, resulting in muscle paralysis. In the “rescued” state, the binding of guanidinium (blue and yellow spheres) effectively closes the channel and blocks sodium leakage. The red sphere represents the location of the disease-causing mutation. The side-chain sticks represent the voltage sensors of the sodium channel.

Open and shut: pain signals in nerve cells

Our daily function depends on signals traveling between nerve cells (neurons) along fine-tuned pathways. Central nervous system neurons contain acid-sensing ion channel 1a (ASIC1a), a protein important in sensing pain and forming memories of fear. An ion channel lodged in the cell membrane that provides a pathway for sodium ions to enter the cell, ASIC1a opens and closes in response to changes in extracellular proton concentrations. When protons accumulate outside the neuron, the channel opens, allowing sodium ions to flow into the cell, depolarizing the cell membrane and generating an electrical signal. The channel eventually becomes desensitized to protons and the gate closes. Scientists have visualized both the open and desensitized channel structures, but the third structure, which forms when the protons dissipate and the channel closes, remained elusive. Using protein crystallography at the ALS, researchers finally visualized the closed channel.

>Read more on the Advanced Light Source website

Animation: As the proton concentration increases or decreases, the gated channel ASIC1a toggles between open and closed positions, controlling the timing of signals traveling through the cell membrane of one neuron en route to the next.

How legionella manipulates the host cell by means of molecular mimics

Using synchrotron light, researchers from CIC bioGUNE have solved the structure of RavN, a protein that Legionella pneumophila uses for stealing functions and resources of the host cell.

Mimicry is the ability of some animals to resemble others in their environment to ensure their survival. A classic example is the stick bug whose shape and colour make him unnoticed to possible predators. Many intracellular pathogens also use molecular mimicry to ensure their survival. A part of a protein of the pathogen resembles another protein totally different from the host and many intracellular microorganisms use this capability to interfere in cellular processes that enable their survival and replication.

The Membrane Trafficking laboratory of the CIC bioGUNE in the Basque Country, led by Aitor Hierro, in collaboration with other groups from the National Institutes of Health in the United States, have been working for several years in understanding how the infectious bacterium Legionella pneumhopila interacts with human cells. During this research, experiments have been carried out at the XALOC beamline of the ALBA Synchrotron and I04 beamline of Diamond Light Source (UK). The results enabled scientists to solve the structure of RavN, a protein of L. pneumophila that uses this molecular mimicry to trick the infected cell.

>Read more on the ALBA website

Figure: (extract) Schematic representation of the structure of RavN1-123 as ribbon diagram displayed in two orientations (rotated by 90° along the x axis). Secondary elements are indicated as spirals (helices) or arrows (beta strands), with the RING/U-box motif colored in orange and the C-terminal structure colored in slate. (Full image here)